antitumor responses of invariant natural killer t cells

Review ArticleAntitumor Responses of Invariant Natural Killer T Cells

Jennie B. Altman,1 Adriana D. Benavides,1 Rupali Das,2 and Hamid Bassiri1

1Division of Infectious Diseases, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA2Division of Oncology Research, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA

Correspondence should be addressed to Hamid Bassiri; [email protected]

Received 27 April 2015; Accepted 26 July 2015

Academic Editor: David E. Gilham

Copyright © 2015 Jennie B. Altman et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Natural killer T (NKT) cells are innate-like lymphocytes that were first described in the late 1980s. Since their initial description,numerous studies have collectively shed light on their development and effector function.These studies have highlighted the uniquerequirements for the activation of these lymphocytes and the functional responses that distinguish these cells from other effectorlymphocyte populations such as conventional T cells and NK cells. This body of literature suggests that NKT cells play diversenonredundant roles in a number of disease processes, including the initiation and propagation of airway hyperreactivity, protectionagainst a variety of pathogens, development of autoimmunity, andmediation of allograft responses. In this review, however, we focuson the role of a specific lineage of NKT cells in antitumor immunity. Specifically, we describe the development of invariant NKT(iNKT) cells and the factors that are critical for their acquisition of effector function. Next, we delineate the mechanisms by whichiNKT cells influence andmodulate the activity of other immune cells to directly or indirectly affect tumor growth. Finally, we reviewthe successes and failures of clinical trials employing iNKT cell-based immunotherapies and explore the future prospects for theuse of such strategies.

1. Introduction

Natural killer T (NKT) cells are innate-like lymphocytestypified by coexpression of receptors characteristic of naturalkiller and conventional T cells [1]. As such, murine NKTcells generally bear Ly49 receptors, NKG2 family of receptors,CD94, and NK1.1 (the latter only being expressed in specificstrains, including the commonly used C57BL/6). HumanNKT cells often express similar surface molecules includingCD56, CD161, CD94, NKG2D, and NKG2A. Both humanand mouse NKT cells display a variety of stimulatory andinhibitory T cell-associated receptors and ligands (e.g., CD28and CD154), whose expression depends on the activationstatus of the cell. Finally, both human and murine NKTpopulations include CD4+ and CD4−CD8− (double negative;DN) subpopulations; while CD8+ NKT cells are found inhumans, they are rare in mice [2].

TheT cell receptors (TCRs) expressed byNKTcells recog-nize the conserved and nonpolymorphic MHC class I-likemolecule, CD1d. Unlike classical MHC class I-like molecules,the expression of CD1d is largely restricted to cells of bone

marrow origin including antigen presenting cells (APCs)such as dendritic cells (DCs), macrophages, and B cells.Furthermore, the CD1d molecule (via heterodimerizationwith 𝛽

2-microglobulin) specializes in displaying lipid moi-

eties rather than protein polypeptides. Importantly, intactexpression of CD1d is critical for the development of NKTcell populations, asCd1d−/−mice are devoid of these cells [3].NKT cells are further subclassified into Type I or II lineages,depending on the composition of their TCR and the CD1d-presented glycolipid antigens to which they respond. TypeI or invariant NKT (iNKT) cells express canonical TCR𝛼chains comprised of specific gene segments (V𝛼14-J𝛼18 inmice and V𝛼24-J𝛼18 in humans) that preferentially pair withspecific TCR𝛽 chains (V𝛽8, V𝛽7, or V𝛽2 in mice and V𝛽11 inhumans).These invariant TCR𝛼𝛽pairings confer reactivity toCD1d and a restricted array of presented glycolipid antigens.The dependence of iNKT cells on the V𝛼14-J𝛼18-comprisedTCR𝛼 is demonstrated by V𝛼14 TCR transgenic mice, inwhich a higher frequency and number of iNKT cells areobserved [4], and also J𝛼18−/− mice, in which no matureiNKT cells develop [5]. Despite the conserved use of the

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2015, Article ID 652875, 10 pageshttp://dx.doi.org/10.1155/2015/652875

2 Journal of Immunology Research

invariant TCR, iNKT cell populations are phenotypically(e.g., presence or absence of CD4 expression) and function-ally (e.g., preferential production of certain cytokines, such asIL-17) diverse.

The prototypical (and first discovered) iNKT cell stimu-latory glycolipid, alpha-galactosylceramide (𝛼-GalCer), wasidentified during a screening for compounds from marinesponges (Agelas species) with antitumor activity [6]. Sincethis initial discovery, a number of naturally occurring andsynthetic lipid antigens have been described to bind CD1dand activate iNKT cells. These cells are now typically iden-tified using CD1d tetramers loaded with 𝛼-GalCer or itssynthetic analogs (e.g., PBS-57; [7]). In contrast, Type II orvariant NKT (vNKT) cells bear a more diverse array of TCR𝛼and 𝛽 chains and have been shown to recognize sulfatidemoieties presented by CD1d [8]. More recently, Type II NKTcells have also begun to be better characterized throughdevelopment of CD1d tetramers loaded with sulfatide [9, 10],but these cells are still less well characterized than theirinvariant brethren. Given that far more is known regardingthe antitumor activity of iNKT cells, we will predominantlyfocus our attention on these cells.

2. iNKT Cell Development and Acquisition ofEffector Function

iNKT cells develop in the thymus, by originating fromCD4+CD8+ double positive (DP) thymocytes. Positive selec-tion of iNKT cells is mediated by homotypic interactionsof DP cells and recognition of glycolipid antigen-CD1dcomplexes [11–14]; however, the nature of the self-antigensinvolved in this process remains somewhat elusive. Likeconventional T cells, maturation of iNKT cells at the DPstage and beyond depends on the ability to construct afunctional TCR and intact signaling. As such, iNKT cellsare profoundly diminished or absent in mice lacking expres-sion of RAG, CD3𝜁, Lck, ZAP-70, SLP-76, ITK, LAT, orVav [15–21]. Transcriptionally, development of iNKT cellsat the DP stage is regulated by the transcription factorROR𝛾t, which prolongs the survival of DP thymocytes byupregulating Bcl-XL, to allow sufficient time for distal TCR𝛼gene segment rearrangements to occur [22, 23]. More recentstudies have shown that HEB, the E protein family of basichelix-loop-helix transcription factors, regulates iNKT celldevelopment by regulating ROR𝛾t and Bcl-XL mRNA [24].Finally, the absence of the transcription factor Runx1 alsoblocks iNKT cell development at the earliest detectable iNKTcell-committed subset [23].

iNKT cell development at the DP stage also criticallydepends on the signals generated by engagement of theSignaling LymphocyteActivationMolecule (SLAM) family ofsurface receptors, which are expressed on developing iNKTcells, as well as conventional DP thymocytes. SLAM familyreceptor signaling is transduced by the adaptor moleculeSAP (SLAM-associated protein), which in turn binds tothe tyrosine kinase Fyn, and results in propagation of aphosphorylation signal [25, 26]. Accordingly, iNKT cells failto develop in mice and humans bearing mutations in the

gene that encodes for SAP [27–29], in mice lacking Fyn orexpressing a mutant version of SAP that cannot bind Fyn[23, 30], in mice in which both Ly108 and SLAM signalingare simultaneously disrupted [31], and in those lacking thetranscription factor cmyb (which is necessary for appropriateexpression of SAP and certain SLAM family members) [32].Taken together, these studies establish the importance of theSLAM-SAP-Fyn signaling axis in iNKT cell development.

Following positive selection, iNKT cells undergo dis-tinct stages of maturation that are characterized by thesequential acquisition of CD24, CD44, and NK1.1:CD24hiCD44loNK1.1− (Stage 0), CD24loCD44loNK1.1−

(Stage 1), CD24loCD44hiNK1.1− (Stage 2), and finallyCD24loCD44hiNK1.1+ (Stage 3) [33]. As these cells progressthrough these developmental stages, they begin to upregulateNK cell markers (e.g., NKG2D and Ly49 receptors), CD69,and CD122 and acquire distinct effector functions (e.g.,production of IL-4, IFN-𝛾, perforin, and granzymes)[34]. Acquisition of these effector functions is tightly reg-ulated by several transcription factors [35]. One of the keyregulators of iNKT cell development and acquisition of aneffector/memory phenotype and functions is the broad com-plex tramtrack bric-a-brac-zinc finger transcription fac-tor PLZF, whose expression is highest in Stage 0 and 1populations [36, 37]. PLZF-deficient animals exhibit a severereduction in iNKT cell number and PLZF-deficient iNKTcells fail to cosecreteTh1 andTh2 cytokines upon stimulation[36, 37]. Recently, it was demonstrated that the lethal-7microRNA posttranscriptionally regulate PLZF expressionand iNKT cell effector functions [38].

The transcription factor T-bet is indispensable for thefinal maturation stages of iNKT cells [39, 40] and absence ofthis transcription factor results in reduced iNKT cell numbersdue to developmental blockade at Stage 2. T-bet-deficientiNKT cells fail to proliferate in response to IL-15 as theylack surface expression of CD122, a component of the IL-15receptor [40]. In addition, T-bet-deficient iNKT cells fail toproduce IFN-𝛾 in response to TCR stimulation and exhibitdefective cytolytic activity [39, 40] as T-bet directly regulatesthe activation of genes associated with mature iNKT cellfunctions, such as perforin, CD178, and IFN-𝛾 [40].

As iNKT cells progress to Stage 1, a proportion of cellsdownregulate CD4, giving rise to DN iNKT cells. Generationof the CD4+ iNKT cell lineage and production of Th2-typecytokines is critically regulated by the transcription factorGATA-3. Similar to PLZF-deficient iNKT cells, GATA-3deficient iNKT cells fail to produce Th1 or Th2 cytokines inresponse to 𝛼-GalCer [41]. Recent studies have identifieda unique subpopulation of NK1.1−CD4− iNKT cells thatare transcriptionally regulated by ROR𝛾t and capable ofproducing large quantities of IL-17 upon stimulation [42].As such, iNKT cells are also sometimes classified into NKT1,NKT2, and NKT17 based on their cytokine production pro-files and respective expression of T-bet, GATA-3, and ROR𝛾t[43, 44]. Finally, mechanistic target of rapamycin (mTOR)signaling has also been shown to be important for iNKT celllineage diversification and acquisition of effector functions[45–48], and loss ofmTOR2may result in loss of NKT17 cells.

Journal of Immunology Research 3

Taken together, these recent studies provide new insightsinto the transcriptional regulation of iNKT cell maturationand functional differentiation.

3. iNKT Cells and Antitumor Immunity

The importance of iNKT cells inmediating protection againsttumors is highlighted by several findings. First, a number ofindependent studies have shown a decrease in the number ofiNKT cells in the peripheral blood of patients with a varietyof cancers and even precancerousmyelodysplastic syndromes[49–51]. Moreover, the iNKT cells that persist appear tohave decreased proliferative and functional responses [52–54]. Interestingly, an increased frequency of peripheral bloodiNKT cells in cancer patients portends a more favorableresponse to therapy [55, 56]. While these observations iden-tify an association between iNKT cell numbers and/or func-tion and development of malignancy, they do not providea direct causal link. This link has been established in anumber ofmouse studies in which the biology of the host andinitiation of tumors can be more systematically manipulatedvia gene knockouts, antibody depletion strategies, and adop-tive transfer of various lymphocyte populations into cancer-predisposed or tumor-challenged hosts.

In mice that are prone to development of tumors due toloss of one allele of a tumor suppressor (p53+/−), absenceof iNKT cells (by virtue of genetic knockout of the J𝛼18gene segment or CD1d) results in earlier and more frequentdevelopment of tumors and thus shorter survival [57], whencompared to iNKT-sufficient littermates. Similarly, treatmentof Cd1d−/− and J𝛼18−/− mice with a carcinogen resulted inincreased incidence and earlier onset of tumors in compari-son to treatedwild typemice [58]. Conversely, administrationof 𝛼-GalCer to mice controlled the growth and metastasisof adoptively transferred [59, 60] or carcinogen-induced[61, 62] or spontaneous [63] tumors. Moreover, adoptivetransfer of iNKT cells into J𝛼18−/− iNKT cell-deficient miceprevented the growth of subcutaneous sarcomas [62]. Finally,adoptive transfer of small numbers of purified iNKT cells intolymphocyte-deficient NOD-Scid-IL2r𝛾−/− (NSG) mice wassufficient to protect mice from challenge with a CD1d+ tumor[64]. These findings collectively argue that iNKT cells playa central and nonredundant role in the response to tumors.Further studieswould shed light on themechanisms bywhichiNKT cells exert these antitumor effects.

3.1. Indirect Cytokine-Mediated Modulation of AntitumorResponses. Engagement of the invariant TCR by CD1d/gly-colipid antigen complexes results in iNKT cell activation,an event that is typified by rapid and robust productionof a variety of cytokines and chemokines, including—butnot limited to—IL-2, IL-4, IL-10, IL-13 IL-17, IFN-𝛾, TNF𝛼,TGF𝛽, GM-CSF, RANTES, eotaxin, MIP-1𝛼, and MIP-1𝛽[65, 66].The nature andmagnitude of the iNKT cell cytokineresponse depend on the glycolipid antigen; for example, 𝛼-GalCer-mediated iNKT cell activation elicits a strong IFN-𝛾-dominated cytokine response, while OCH (a syntheticanalog of 𝛼-GalCer with a truncated lipid chain) elicits

a response with significantly higher level of IL-4 production[67]. The rapidity of this cytokine response is attributedto the semiactivated state of iNKT cells and the presenceof preformed cytosolic mRNA for a variety of cytokines[68]. Indeed, administration of 𝛼-GalCer to iNKT cell-sufficient, but not iNKT cell-deficient, mice results in poly-clonal activation of conventional T, B, and NK cells within3-4 hours [69] and also eventually leads to the mobilizationof macrophages and neutrophils [70]. Intriguingly, it waspreviously believed that mammalian species are incapable ofproducing glycolipids (such as 𝛼-GalCer), in which the sugarmoiety is attached via anO-linkage to the ceramide backbonein an alpha-anomeric configuration. Despite the absence of𝛼-glucosyl or 𝛼-galactosyl transferases in mammals, recentfindings indicate that a small percentage of the glycolipidsthat are constitutively presented by mammalian CD1d areindeed 𝛼-anomeric [71]. Whether the percentage of CD1d-presented 𝛼-anomeric glycolipids is altered in tumor tissuesrepresents an interesting question that deserves further futureinvestigation.

Nonetheless, following encounter with CD1d/antigencomplexes displayed by APCs, iNKT cells not only producecytokines but also upregulate surface expression of CD154(see Figure 1(a)). Ligation of APC-expressed CD40 is espe-cially important for mediating subsequent maturation andfunctional activation of DCs, subsequent upregulation ofCD80 and CD86, and amplified production of IFN-𝛾 [72, 73].In addition, the ligation of the chemokine receptor CXCR6on iNKT cells by CXCL16 expressed on APCs also providescostimulatory signals resulting in robust 𝛼-GalCer-inducediNKT cell activation [74]. Importantly, matured DCs arepotent producers of IL-12, which induces sustained IFN-𝛾 production by iNKT cells [75–77]. The importance ofiNKT cells in IL-12-mediated tumor rejection was effectivelydemonstrated by the defective clearance of a variety of tumorsin J𝛼18−/− mice [5]. Mature DCs also support the primingand activation of CD8+ T cells, culminating in optimaleffector and memory cell formation [72, 78]. Finally, thesustained release of IFN-𝛾 by iNKT cells leads to activationand proliferation of NK cells and NK cell secretion of IFN-𝛾.The combination of cytokines (e.g., IL-2, IL-12, and IFN-𝛾) asa result of iNKT cell activation also leads to upregulation ofdeath-inducing ligands (e.g., CD178 or CD253) on NK cellsand CD8+ T cells [79, 80]. These sequential activation eventsare believed to be critical for the 𝛼-GalCer-induced iNKTcell-mediated antitumor effects [76, 81, 82]. As such, iNKTcells not only bridge the activation of innate and adaptiveimmunity, but also indirectly potentiate the antitumor activ-ity of other cytotoxic effector lymphocytes.

3.2. Indirect Control of Tumor Growth via Alteration ofTumor Microenvironment. Tumor establishment and growthare believed to be intricately modulated by a myriad ofsoluble and contact-derived signals obtained from the tumormicroenvironment (TME), which consists of the tumorcells themselves, tumor-infiltrating lymphocytes (TILs), andstromal cells that communicate in a dynamic and bidirec-tional manner. In addition to their indirect modulation of


TCRGAg

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perforin andgranzymeexocytosis

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(a) (b) (c)

Figure 1: Possible mechanisms of iNKT cell-mediated antitumor responses. (a) Indirect cytotoxicity. iNKT cells and DCs reciprocallycoactivate each other via TCR:CD1d/GAg and CD40:CD154 interactions, resulting in the release of several cytokines that secondarily activateand promote the antitumor cytotoxicity of other effector lymphocytes. (b) Modulation of the TME. iNKT cells kill tumor-supporting cells,such as TAMs andMDSCs, and also limit angiogenesis, to indirectly control tumor growth. (c) Direct cytotoxicity. iNKT cells mediate lysis oftumor targets via engagement of TCR or NKG2D. Lightning bolt: exertion of direct cytotoxicity; DC: dendritic cell; GAg: glycolipid antigen;TC: tumor cell; TAM: tumor-associated macrophage; MDSC: myeloid-derived suppressor cell; TME: tumor microenvironment.

other effector lymphocyte populations, iNKT cells may alsoregulate tumor growth via their effects on the TME (seeFigure 1(b)). Indeed following intravenous administration,iNKT cells were shown to represent a significant percentageof the TILs in patients with head and neck carcinomas[83, 84]. Importantly, higher frequency of tumor-infiltratingiNKT cells correlated with overall and disease-free survivalas an independent prognostic factor in primary colorectalcancer patients [85] and with tumor regression in headand neck carcinomas [86]. Conversely, in patients withprimary hepatocellular or metastatic cancer, CD4+ iNKTcells that produced high levels of Th2-type cytokines andhad low cytolytic activity were enriched within the tumorand appeared to inhibit the expansion of antigen-specificCD8+ T cells, suggesting that these particular iNKT cells maycontribute to generate an immunosuppressive microenviron-ment [86].

In experimental studies, cotransfer of human mono-cytes and iNKT cells to tumor-bearing NOD-Scid micesuppressed tumor growth when compared with mice thatreceived monocytes alone [87]. Importantly, iNKT cellscan target tumor supportive cells such as tumor-associatedmacrophages (TAMs), a highly plastic monocyte-derived

subset of inflammatory cells that can exert immunosuppres-sive functions, and promote tumor proliferation and matrixturnover [88, 89]. Indeed TAMs are known to produce IL-6, a cytokine that appears to promote the proliferation ofmany solid tumors, including neuroblastomas and breastand prostate carcinomas [87]. Consistent with the tumor-permissive capacities of TAMs, Chen et al. found thatmacrophage density correlated positively with microvesselcounts and negatively with patient relapse-free survival [90].Since TAMs cross-present neuroblastomaderived endoge-nous CD1d ligand(s), they can be specifically recognized andkilled by iNKT cells in an IL-15-dependent process [87].Other potential iNKT cell TME targets include myeloid-derived suppressor cells (MDSCs). MDSCs have been foundto accumulate in the blood, lymph nodes, and bone marrowand at tumor sites in most patients and experimental animalswith cancer and inhibit both adaptive and innate immunity[91]. The absence of iNKT cells in mice during influenzavirus infection resulted in the expansion of MDSCs, highviral titer, and increased mortality. The adoptive transferof iNKT cells abolished the suppressive activity of MDSCsand restored virus-specific immune responses, resulting inreduced viral titers and increased rates of host survival [92].


Thus, certain populations of iNKT cells may help alter theTME via their effects on TAMs and MDSCs, to help createa tumor-suppressive or immune-permissive milieu.

3.3. Direct Antitumor Cytotoxicity. In addition to their indi-rect control of tumor growth, iNKT cells can mediate directkilling of tumor targets (see Figure 1(c)). iNKT cells alone,or in combination with NK cells, have been shown tokill a variety of tumor targets in vitro [6, 93, 94]. Whilethis mechanism of killing appears to be dependent on thepresence of stimulatory glycolipids and CD1d [95, 96], iNKTcell cytotoxicity also appears to be triggered via ligationof NKG2D by target-expressed stress ligands [97]. NKG2Dligation can also costimulate TCR-triggered cytotoxicity [97].It remains to be seen whether MULT1, the newly identifiedshed form of high affinity NKG2D ligand that triggers NK-mediated tumor rejection in mice, also activates iNKT cells[98].

Consistent with their direct cytotoxic capacity, iNKTcells express perforin and granzymes, as well as CD178[34, 96, 99, 100]. In our hands, blockade of CD1d-mediatedlipid antigen presentation, disruption of T cell receptor(TCR) signaling, or loss of perforin expression was found tosignificantly reduce iNKT cell killing in vitro [64]. Moreover,we demonstrated that iNKT cells alone were sufficient forcontrol of the growth of a T cell lymphoma in vivo thatpreferentially relies on perforin and the adaptor proteinSAP [64, 69]. Mechanistically, iNKT cells rely on SAP forformation of stable conjugates with the tumor targets aswell as proper orientation of the lytic machinery at theimmunological synapse [69]. Despite the majority of studiesimplicating iNKT cells as having an antitumor role, a limitednumber of studies also implicate iNKT cells as suppressingantitumor responses [101], but these paradoxic responsesmay be related to the level of tumor CD1d expression [102,103]. Alternatively, these differences may stem from the factthat—contrary to the use of C57BL/6 mice in the previouslydiscussed studies—these last two studies were performed inBALB/c mice, in which there is a predominance of IL-4-producingTh2 phenotype iNKT cells [43].

Interestingly, the antitumor responses of iNKT cells maybe regulated by the activity of Type II NKT cells [104]. Terabeet al. demonstrated that Type II variant NKT (vNKT) cellswere sufficient for the downregulation of tumor immuno-surveillance and relapse growth of a model fibrosarcoma inan antigen-dependent manner [105], while a second studyfound that activation of vNKT cells with sulfatide antigencould suppress the activation of iNKT cells [106]. Thesesuppressive vNKT cells were found to be predominantlyCD4+ [107]. Conversely, Type II vNKT cells were, in at leastone study, suggested to promote the antitumor activity ofCpG oligodeoxynucleotides [108].

iNKT cell antitumor activity is also suppressed by regu-latory T (Treg) cells. This suppression appears to be medi-ated through a contact- and IL-10-dependent mechanism[109, 110]. Indeed, induction of Treg cells suppressed theprotective effect of adoptive transfer of iNKT cells intoJ𝛼18−/−mice [111]. Consistent with these findings, depletion

of Treg cells or short-term elimination of their suppressiveactivity results in enhanced iNKT cell-mediated antitumorresponses and increased NK and CD8 T cell activation andIFN-𝛾 production [112]. Interestingly, the ability of Treg cellsto suppress iNKT cell proliferation depends on the degreeof invariant TCR agonism, such that responses to weak(e.g., OCH), but not strong (e.g., 𝛼-GalCer), agonists wereeffectively suppressed [110]. When viewed collectively, thesefindings suggest that iNKT cells possess inherent capacity fordirect cytotoxicity but their antigenic exposuremaymodulatewhether their antitumor effects can be suppressed byTreg andvNKT cells.

4. iNKT Cell-Based Immunotherapy

Given the preponderance of evidence suggesting that theactivation of iNKT cells provides protection against thegrowth and metastasis of a variety of tumors, safety of𝛼-GalCer administration was examined in a Phase I trial[113]. While administration of 𝛼-GalCer was well toleratedat a range of doses, no clinical responses were observed inpatients with advanced solid tumors. On the heels of thisstudy, Nieda et al. showed that treatment of metastatic cancerpatients with 𝛼-GalCer-pulsed immature monocyte-derivedDCs resulted in dramatic increases in serum IFN-𝛾 and IL-12and activation of NK and T cells in the majority of subjects.Importantly, this Phase I trial also documented reductionin tumor biomarkers and tumor necrosis in several patients[100]. These findings were extended in a study of a smallnumber of patients, in which the 𝛼-GalCer-pulsed DCs werematured prior to adoptive transfer.This study demonstrated a>100-fold increase in blood iNKT cell numbers in all patients,and this increase was long-lived (>6months) [114]. A numberof subsequent clinical trials, all with limited number ofpatients with advanced head and neck or non-small cell lungcancers, have since employed similar strategies of adoptivetransfer of 𝛼-GalCer-pulsed APCs [115–118]. Collectively,these studies demonstrate increases in blood IFN-𝛾 levels andiNKT cells in some but not all patients, stabilization of diseasein a few of the subjects, and absence of severe treatment-related toxicities.

In a different approach, chemotherapy-refractory 5 lym-phoma patients were treated with autologous peripheralblood mononuclear cells (PBMCs) stimulated with anti-CD3, IL-2, and IFN-𝛾. This ex vivo stimulation resulted inenrichment of NKT cells (to ∼20% on average), and this cellfraction was shown to possess the highest cytotoxic capacityin vitro. Of the nine patients who received adoptive transferof these cells, two showed partial responses and two othershad stabilization of disease [119]. Two subsequent studies byMotohashi et al. evaluated the adoptive transfer of ex vivoexpanded iNKT cell-enriched cells to patients with advancedcancer. In the first, 6 patients with advanced non-small celllung cancer were treated with either a low or a high dose ofex vivo expanded iNKT cells. Of the 3 patients treated withthe high dose, all had an increase in the frequency of IFN-𝛾-producing PBMCs and 2 showed expansion of iNKT cells[120]. Although no clinical responses were observed in this


study, a follow-up trial of 17 patients with advanced headand neck cancers treated with a high dose of iNKT cell-enriched autologous PBMCs showed a significant increase inIFN-𝛾-producing PBMCs in 10 of 17 patients. Importantly,while none of these patients displayed tumor regression, 5had disease stabilization and the mean survival time for thesubjects with higher frequencies of IFN-𝛾-producing PBMCswas tripled above those with low percentages of IFN-𝛾-producing PBMCs (29.3 versus 9.7 months) [117]. Finally, in acombinational treatment strategy, Kunii et al. administeredboth in vitro expanded iNKT cells and 𝛼-GalCer-pulsedAPCs to patients with advanced head and neck squamouscell carcinomas. Treatment increased the frequencies of iNKTcells and IFN-𝛾-producing PBMCs, and a partial clinicalresponse or disease stabilization was observed in 7 of 8patients [83]. Although the responses in these studies havenot been profound, it must be noted that these iNKT cell-based immunotherapies have all been conducted on patientswith advanced malignancies—often those in whom standardchemotherapy, irradiation, and/or surgical excision treat-ments had failed.Moreover, themajority of these patients hadCD1d− cancers.

Future studies of iNKT cell-based immunotherapy maybe able to take advantage of two recent technologies. Asmentioned previously,manymalignancies are associatedwitha decrease in the numbers and proliferative capacity ofperipheral blood iNKT cells. In order to circumvent thedifficulty of being able to expand these infrequent and poten-tially defective cells from patients, Watarai et al. generatedinduced pluripotent stem (iPS) cells from mature iNKTcells and then expanded large numbers of iNKT cells fromthese established iPS cells. iPS-NKT cells generated in thisfashion were demonstrated to be able to activate and expandantigen-specific CD8 T cell responses to limit the growthof leukemia in mice [121] without inducing graft versushost disease (GvHD) [122]. The second strategy employschimeric antigen receptors (CARs). Recently, a report byHeczey et al. described iNKT cells engineered to expressCARs bearing specificity for GD2, a highly expressed moietyon neuroblastoma cells. In their studies, they showed thatiNKT cells expanded from the PBMCs of healthy humandonors and transduced with retroviral CAR constructs couldprotect humanized NSG mice against metastatic neurob-lastoma without inducing GvHD [123]. Whether these twotechnologies could be combined to generate functional CAR-bearing iPS-NKT cells remains to be seen.

5. Concluding Remarks

iNKT cells are innate-like effector lymphocytes that notonly are directly cytotoxic, but also possess the uniqueability to nucleate the antitumor responses of other effectorlymphocytes and alter the cellular and angiogenic makeupof the tumor microenvironment. As such, the promise of aneffective iNKT cell-based immunotherapy can only be real-ized by devising and evaluating strategies that simultaneouslymaximize each of these antitumor effector mechanisms. Thechallenge for the futurewill thus be to identify these strategies

and apply them to tumors against which iNKT cells wield themost optimal responses.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

Acknowledgments

The authors wish to acknowledge past and current grantsupport for RupaliDas byAlex’s Lemonade Stand Foundationand for Hamid Bassiri by Clinical Immunology Society &Talecris Biotherapeutics, American Cancer Society (IRG-78-002-36) and the National Institutes of Health (T32-AI007634,K12-HD04335, and K08-CA166184). In addition, the authorswish to thank Ashlyn E. Bassiri for critical appraisal of thispaper, Charles H. Pletcher at the University of PennsylvaniaFlow Cytometry Core for technical advice, and the NIHTetramer Core Facility for their gracious support.

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